Cells were cultured for 12 h in 6-well plates at a density of 1 × 106 cells/well in the presence or absence of 8-nitro-cGMP (30 and 100 μmol/L). Cells were washed two times with PBS and collected using a scraper, then lysed in RIPA buffer (Fujifilm Wako Pure Chemical Co.). Cell lysates (20 μg of protein) were subjected to SDS-PAGE (10% acrylamide) under reducing conditions, then separated proteins were transferred onto PVDF membranes and immunoblotted using an anti-RS-cGMP antibody(10 (link)) and β-actin. Immunoreactive bands were visualized by an enhanced chemiluminescence reaction with an ECL Prime Western Blot Detection System (GE Healthcare, Chicago, IL). The intensity of whole bands detected by anti-RS-cGMP antibody and that by anti-β-actin antibody was quantified using Versa Doc 5000 MP (BioRad Laboratories, Hercules, CA).
Ecl prime western blot detection system
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The ECL Prime Western Blot Detection System is a laboratory equipment used to detect and visualize proteins in Western blot analysis. It utilizes an enhanced chemiluminescent (ECL) reaction to generate a luminescent signal proportional to the amount of target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated donkey anti rabbit igg, by GE Healthcare (3 mentions)
Versadoc 5000mp, by Bio-Rad (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Fujifilm (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Foxm1, by Santa Cruz Biotechnology (1 mentions)
Claudin 1, by Cell Signaling Technology (1 mentions)
Cell fractionation kit, by Cell Signaling Technology (1 mentions)
Lysis buffer, by Agilent Technologies (1 mentions)
Histone h3 d1h2 xp, by Cell Signaling Technology (1 mentions)
Criterion tgx gel, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Rabbit monoclonal antibodies, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Dc protein assay reagent, by Bio-Rad (1 mentions)
Ha hrp, by Roche (1 mentions)
Ha hrp, by Merck Group (1 mentions)
11 protocols using ecl prime western blot detection system
1
Quantifying 8-nitro-cGMP Effects on Protein Levels
2
Protein Expression Analysis by Western Blot
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Cell lysis, SDS-polyacrylamide gel electrophoresis and Western blottings were performed as previously described [13 (link)]. The immunoreaction was developed using the ECL prime Western blot detection system (GE Healthcare). The images of ECL signals were taken and analyzed using the Amersham Imager600 (AI600) and AI600 analysis software (GE Healthcare). The specific primary antibodies for Western blot analysis were 1:10,000 anti-β-actin (A5441, Sigma Aldrich, St. Louis, MO), 1:200 FOXM1 (sc-502 and sc-376471; Santa Cruz Biotechnology, Santa Cruz, CA), and 1:1,000 claudin-1 (13255S; Cell Signaling Technology; Denvers, MA).
3
Analyzing Glucocorticoid Receptor Isoforms
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tsA201 cells transfected with recombinant hGR isoforms were either lysed in ice-cold lysis buffer (Agilent) supplemented with cOmplete Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and supernatants harvested and normalized, or were fractionated into nuclear, cytoplasmic, and membrane extracts using a cell fractionation kit (Cell Signaling Technology, Danvers, MA). Proteins were run on a 4–20% BioRad Criterion TGX gel (Hercules, CA), then transferred to a polyvinylidene difluoride membrane (BioRad). Membranes were blocked with 5% nonfat dry milk, washed, and incubated overnight with Glucocorticoid Receptor (D8H2) XP (1:1000), MEK1/2 (D1A5) (1:1000), AIF (D39D2) XP (1:1000), or Histone H3 (D1H2) XP (1:2000) rabbit monoclonal antibodies (Cell Signaling Technology) in 5% nonfat milk or bovine serum albumin. A secondary anti-rabbit-HRP (GE Healthcare, Piscataway, NJ) in 5% nonfat milk at 1:2000 was used for protein visualization via chemiluminescence using the ECL Prime Western Blot Detection System (GE Healthcare).
4
Quantitative Western Blot Analysis of Protein Extracts
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Western blot analysis was performed in accordance with standard protocols [96 ]. For whole-cell protein extract preparation, 1.5 × 106 cells were pelleted by centrifugation at 1250g and lysed in 100 μl of Laemmli sample buffer (Bio-Rad). Protein concentrations were determined using the Bio-Rad DC protein assay reagent in accordance with the manufacturer’s protocol. 15–20 μg of whole-cell protein extracts, 10 μl of nuclear extracts, or entire eluate from immunoprecipitated samples were separated on an 8 % SDS–polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Thermo Scientific) and subjected to western blot analysis. Membranes were probed with various rabbit primary antibodies followed by incubation with donkey anti-rabbit HRP-conjugated IgG (1:3000; GE Healthcare, NA9340) secondary antibody where applicable. The antibody signals were detected using the ECL+ or ECL prime western blot detection system (GE Healthcare). Primary antibodies used: HA-HRP (1:6000; Roche, 2013189), SIN3 (1:2000; [21 (link)]), RPD3 (1:3000; [21 (link)]), dKDM5/LID (1:5000; kindly provided by Dr. Julie Secombe [32 (link)]), beta-actin (1:1000; Cell Signaling, 4967).
5
Western Blot for Protein Detection
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Western blot was carried out following standard protocols [52 ]. To prepare whole cell extract, approximately 1.5x106 cells were harvested by centrifugation and lysed in Laemmli Sample buffer (Bio-Rad). Proteins (15 to 20 μg) were resolved on an 8 % SDS-polyacrylamide gel and transferred to a polyvinylidine difluoride (PDVF) membrane (Pall). Membranes were incubated with primary antibody: HA-HRP (1:6000; Sigma), SIN3 PAN (1:2000 [40 (link)]), SIN3 220 (1:2000 [35 (link)]), α-Tubulin (1:1000, Cell signaling) or β-Actin (1:1000, Cell signaling). Following incubation with primary antibody, membranes were incubated with donkey anti-rabbit HRP-conjugated IgG (1:3000, GE healthcare) secondary antibody wherever applicable. The antibody signal was detected using the ECL prime western blot detection system (GE Healthcare).
6
Western Blot Protein Detection
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Cells were washed three times using PBS and suspended for 30 min on ice in lysis buffer (1% Nonidet P40, 10% glycerol, 1 mM CaCl2 in HEPES/NaCl) supplemented with 1% protease inhibitor (Sigma-Aldrich #P8340, Burlington, MA, USA). Supernatants were collected after centrifugation and total protein concentration determined by Bradford assay. Then, 25 µg of total protein was separated in reducing conditions by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad) followed by blocking with 5% skimmed milk in PBS supplemented with 0.5% Tween 20 (PBS-T). The membranes were incubated with their respective primary antibody for one hour at room temperature. After washing three times using PBS-T, the membranes were incubated with the indicated horse radish peroxidase (HRP)-conjugated secondary antibodies. Following extensive washing, protein levels were detected using ECL Prime Western blot detection system (GE Healthcare, Chicago, IL, USA) and visualized using the ChemoStar Professional Imager System (Intas, Göttingen, Germany).
7
Extracellular Vesicle Protein Analysis
8
Western Blot Analysis of Immune Signaling Proteins
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The cells were washed with PBS and scraped using a cell scraper. The cells were transferred to 1.5 mL microtubes and centrifuged at 5000 g for 3 min. Cells were lysed in 100 μL of lysis buffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 6% 2‐mercaptoethanol, 10% glycerol, 0.0125% bromothymol blue) and heated at 95°C for 5 min. Whole cell extracts were subjected to electrophoresis on 4%–20% Mini PROTEAN TGX gels (Bio‐Rad, 4561095) and then transferred to Trans‐Blot Turbo Transfer Pack membranes (0.2 μM PVDF, BIO‐RAD, 1704156). Membranes were blocked with 5% BSA in Tris‐Buffered Saline‐Tween, TBST 0.001% azide for 60 min and probed with primary anti‐STING (D2P2F) rabbit mAb (#13647, Cell Signaling Technology), anti‐cGAS (D1D3G) rabbit mAb (#15102, Cell Signaling Technology), anti‐EGF‐Receptor (D38B1) XP rabbit mAb (#4267), and anti‐β‐actin (13E5) rabbit mAb (#4970, Cell Signaling Technology). The membranes were then washed with TBST and incubated with a secondary donkey anti‐rabbit immunoglobulin antibody conjugated to horseradish peroxidase (HRP) (Abcam, ab205722) for 1 h. Proteins were detected using a chemiluminescence detection system (ECL Prime Western Blot Detection System; GE Healthcare, RPN2232) according to the manufacturer's instructions.
9
Western Blot Analysis of Histone Modifications
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Western blot analysis of whole cell protein extract was performed in accordance with standard protocols as described previously (Pile et al. 2002 (link)). Whole cell protein extracts (12 μg) were separated on a 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Pall). Membranes were probed with various rabbit primary antibodies followed by incubation with donkey anti-rabbit HRP-conjugated IgG (1:3000; GE Healthcare) secondary antibody. The antibody signals were detected using the clarity western ECL substrate (Bio-Rad) for H3K4me2 and H3K4me3 or ECL prime western blot detection system (GE Healthcare) for H3K9me2 and H4. Primary antibodies included: H3K4me2 (1:5000; Millipore), H3K4me3 (1:2500; Active Motif), H3K9me2 (1:500; Millipore) and H4 (1:15,000; Abcam) as a loading control. At least three biological replicates were performed.
10
Western Blot Analysis of Signaling Pathways
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C2C12 cells were lysed in 10 mmol/L Tris-HCl (pH 7.8) with 1% Nonidet P-40, 0.15 mol/L NaCl, and a protease inhibitor mixture containing EDTA (Roche Applied Science, Penzberg, Germany). Cell lysates (5 μg of protein) were subjected to SDS-PAGE (10% polyacrylamide gel) under a reducing condition. Following electrophoresis, proteins were transferred onto PVDF membranes and incubated overnight at 4 °C with the primary antibodies against smad1, phospho-smad1/5, ERK1/2, phospho-ERK1/2, ERK5, phospho-ERK5, JNK, phospho-JNK, p38 MAPK, phospho-p38 MAPK, AKT, and phosphor-AKT, followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare, Little Chalfont, UK). Immunoreactive bands were visualized by an enhanced chemiluminescence reaction with an ECL Prime Western Blot Detection System (GE Healthcare). Intensity of the chemiluminescent bands was quantitatively analyzed using Versa Doc 5000 MP (Bio-Rad Laboratories, Hercules, CA, USA). The ratio for intensity of a band for phosphorylated protein to that for total protein was calculated.
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